1. Volume 16, Issue 6, Pages 873-884 (June 2008)
Autoregulation by the Juxtamembrane Region of the Human Ephrin Receptor Tyrosine Kinase A3 (EphA3) 
Tara L. Davis, John R. Walker, Peter Loppnau, Christine Butler-Cole, Abdellah Allali-Hassani, Sirano Dhe-Paganon 
Structure 
Volume 16, Issue 6, Pages (June 2008)
DOI: /j.str
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2. Figure 1
Constructs Used in the Current Study and Alignment of EphA3 with Selected Eph RTKs
(A) Graphical representation of EphA3; domains referenced in the text are labeled and colored. Residues highlighted in the text are also labeled. Below, a graphical representation of the constructs and mutants described in the text.
(B) Alignment was performed using ClustalX (Thompson et al., 1997), and the cytosolic region roughly corresponding to the JMKIN boundaries were used for the alignment. Conserved residues are color-coded according to chemical property if there is at least 90% identity at that position. In addition, identity is marked above the alignment as follows: asterisk (absolute conservation), colon (conservative substitutions), and period (semiconserved substitutions). Secondary structural elements are labeled and shown graphically above the alignment and the positions of the JMS, AL, and the linker region between the kinase and SAM domains are indicated. Numbering corresponds to human EphA3.
Structure  , DOI: ( /j.str )
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3. Figure 2 Overall Structure of EphA3 Kinase
All molecular graphics figures were generated using PyMOL unless noted.
(A) Cartoon representation of the EphA3 JMKIN base ANP structure. The structure is shown in forest green and in cartoon representation. Secondary structure elements and regions discussed in the test are labeled. Regions of disorder are indicated with dashed lines. This structure was chosen because it represents the highest degree of order modeled for all the EphA3 structures.
(B) Ribbon representation of JMKIN base ANP overlaid with EphB2 in pink (1JPA). Alignment over all atoms yields an rmsd of less than 1Å for all structures; key regions of structural difference occur in the N-terminal lobe, centered on the JMS and the AL, along with slight differences in the β1-G loop-β2 region.
Structure  , DOI: ( /j.str )
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4. Figure 3 Tyr701 Is an Autophosphorylation Site in EphA3
The model for the JMKIN base ANP structure is shown in cartoon and stick representation and colored forest green. The 2Fo-Fc density for phosphotyrosine701 (PTR701) and the ANP nucleotide in the active site in the JMKIN base ANP structure is contoured at 0.9σ. An orientation is shown that best reveals the density and position of the PTR701 relative to the nucleotide. Residues contributed by the β1 strand and G loop in the N-terminal lobe and others which line the nucleotide binding site are shown in stick representation.
Structure  , DOI: ( /j.str )
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5. Figure 4
The Linker Between the Kinase and SAM Domains Binds into a Complementary Pocket on the C-terminal Lobe of the Kinase Domain
An electrostatic surface was generated using APBS (Baker et al., 2001) using a gradient from −10 to 10 keT. Shown in cartoon and stick representation is the model for KIN ANP in firebrick red; this model contains the most ordered linker region comprising residues 885–906. Highlighted is the complementary surface made up by Tyr841, Leu901, and Leu903.
Structure  , DOI: ( /j.str )
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6. Figure 5
Rotamer Position of Y742 and S768 in Various Wild-Type EphA3 Structures
The region including the αE-β6 and β7-N-terminal AL region are highlighted in each panel.
(A) JMKIN base ANP. In the ANP-PNP bound untreated structure, shown in forest green, Tyr742 and Tyr596 are in a single and complementary stacking conformation; the AL shows a relatively high degree of disorder, and the conformation of Ser768 is undetermined. This structure is considered an “off” conformation of the enzyme.
(B) The JMKIN base apo structure, in teal, shows the clash between a single rotamer of Ser768 and a single rotamer of Tyr742. Part of the JMS is present, showing the relationship between Tyr742 and JX1 (Tyr596). This structure is considered an intermediate low-activity conformation in our analysis.
(C) JMKIN ATP ANP. In this example of a high-activity conformation, shown in goldenrod, both Ser768 and Tyr742 are in dual rotamer positions. The JMS is completely disordered in this structure.
Structure  , DOI: ( /j.str )
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7. Figure 6 Density of the Ser768:Tyr742 Clash Region
The structure of JMKIN ATP ANP is shown in cartoon and stick representation and colored goldenrod. 2Fo − Fc density for the Ser768:Tyr742 clash region is shown in teal, contoured at 0.9σ. Complete density is observed for both rotamers of Ser768, and nearly complete density is observed for both Tyr742 rotamers.
Structure  , DOI: ( /j.str )
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8. Figure 7 B-Factor Analysis of EphA3 Structures
(A) Shown is a graph representing the greatest variance in B-factors between wild-type EphA3 structures, after normalization of the main chain values against a reference structure. The highlighted region corresponds to the β1–β2 region, which closes down upon the nucleotide and substrate binding pocket upon kinase activation and substrate binding. The normalized B-factor for this region increases in order of increasingly active structure, with the JMS containing and dephosphorylated structure considered inactive and the JMS deleted ANP bound kinase structure considered the most active.
(B) Shown is an overlay of the seven wild-type EphA3 kinase models, colored by B-factor values. The B-factors are separated into 5 bins, colored in increasing order from blue (less than 15 Å2) to red (greater than 31 Å2). Clearly, the N-terminal lobe dominates in terms of B-factor considerations, and the β1-G loop-β2 region is again highlighted with the highest overall B-factors for all structures.
Structure  , DOI: ( /j.str )
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9. Figure 8 Kinase Activity and Structures of EphA3 Mutants
(A) All proteins were purified under identical conditions and pretreated with excess ATP and MgCl2 to fully phosphorylate the enzyme before kinetic analysis as described in the Experimental Procedures. All experiments were performed in duplicate. For error bars, the standard error in Km and kcat were propagated in order to show the resultant error in kcat/Km.
(B) Presented in order of increasing activity are three EphA3 mutants. The YYFF:Y742F model has a disordered AL and ordered JMS region and is nearly inactive compared to wild-type enzyme. The YYFF:S768A mutant has an ordered AL and ordered JMS region and has recovered almost complete activity relative to wild-type enzyme. The YYFF:Y742A mutant has an ordered AL and partially ordered JMS region and is nearly three-fold more catalytically efficient than wild-type enzyme.
Structure  , DOI: ( /j.str )
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